Matrix protein compositions for guided connective tissue growth

ABSTRACT

The present invention relates to the use of enamel matrix, enamel matrix derivatives and/or enamel matrix proteins as therapeutic and/or cosmetic agents. These substances are used for the manufacture of a pharmaceutical and/or cosmetic composition for actively inducing, guiding and/or stimulating connective tissue growth and thus to prevent connective tissue scarring and/or contraction in a wound cavity and/or tissue defect that is characterized by a substantial loss of tissue. The invention comprises, in particular, the use of active enamel substances for guided connective soft tissue growth and resistance to contraction in deep cavity shaped wounds following loss or removal of significant volumes of tissue, such as e.g., after surgical removal of a tumor and especially in combination with radiation therapy.

FIELD OF THE INVENTION

The present invention relates to the use of enamel matrix, enamel matrixderivatives and/or enamel matrix proteins as therapeutic, asprophylactic and/or as cosmetic agents. In the present invention, saidsubstances are shown to actively induce, guide and/or stimulateconnective tissue growth and to be involved in preventing connectivetissue scaring and/or contraction. Comprised in the invention is inparticular the use of active enamel substances for guided connectivesoft tissue growth and resistance to contraction in deep cavity shapedwounds following loss or removal of significant volumes of tissue, suchas e.g. after surgical removal of a tumour and especially in combinationwith radiation therapy.

BACKGROUND OF THE INVENTION

Removal of a significant volume of tissue, as is frequently necessary incancer surgery, confronts the surgeon with special technical andphysiological problems. Removal of a significant volume of tissue oftenmakes it difficult to close the wound properly, and when closing isachieved, the tissue scars and the stitches restrict the mobility, whichdisabilitates the patient. The enclosed wounds can collapse or fill withfluids and cell poor scar tissues. The results are often disabling scartissue formation, infections, pain, cosmetic anomalies or even completeloss of function of the effected body parts. For example, after surgicalremoval of a breast tumour, the contraction of the wound cavity oftenleads to shrinkage of the breast that by far exceeds the volume of theoriginally removed tissue. Furthermore, adjuvant radiation therapyincreases the above mentioned problems drastically. Today, thesepost-surgical conditions are treated by silicon inlays, secondarypalliative surgery, pain and infection controlling drugs, or in theworst case by amputation. Needles to say, these conditions andmeasurements all cause severe problems for the patients, physically,esthetical and emotionally.

Enamel matrix proteins, present in the enamel matrix, are mostwell-known as precursors to enamel. Prior to cementum formation, enamelmatrix proteins are deposited on the root surface at the apical end ofthe developing tooth-root. There is evidence that the deposited enamelmatrix is the initiating factor for the formation of cementum. Again,the formation of cementum in itself is associated with the developmentof the periodontal ligament and the alveolar bone. As shown by thepresent inventors prior to the present invention, enamel matrix proteinscan therefore promote periodontal regeneration through mimicking thenatural attachment development in the tooth (Gestrelius S, LyngstadaasSP, Hammarstrøm L. Emdogain—periodontal regeneration based onbiomimicry. Clin Oral Invest 4:120–125 (2000).

The enamel matrix is composed of a number of proteins, such asamelogenin, enamelin, tuft protein, proteases, and albumin. Amelogenins,the major constituent of the enamel matrix, are a family of hydrophobicproteins derived from a single gene by alternative splicing andcontrolled post secretory processing. They are highly conservedthroughout vertebrate evolution and demonstrate a high overall level ofsequence homology among all higher vertebrates examined (>80%). In fact,the sequences of porcine and human amelogenin gene transcript differonly in 4% of the bases. Thus, enamel matrix proteins, although ofporcine origin, are considered “self” when encountered in the human bodyand can promote dental regeneration in humans without triggeringallergic responses or other undesirable reactions.

Enamel matrix derivative (EMD), in the form of a purified acid extractof proteins from pig enamel matrix has previously been successfullyemployed to restore functional periodontal ligament, cementum andalveolar bone in patients with severe tooth attachment loss (Hammarströmet al., 1997, Journal of Clinical Periodontology 24, 658–668).

In studies on cultured periodontal ligament cells (PDL), it wasfurthermore shown that the attachment rate, growth and metabolism ofthese cells were significantly increased when EMD was present in thecultures. Also, cells exposed to EMD showed increased intracellular cAMPsignalling and autocrine production of growth factors, when compared tocontrols. Epithelial cells on the other hand, increased cAMP signallingand growth factor secretion when EMD was present, but theirproliferation and growth were inhibited (Lyngstadaas et al., 2001,Journal of Clinical Periodontology 28, 181–188).

Enamel proteins and enamel matrix derivatives have previously beendescribed in the patent literature to be able to induce hard tissueformation (i.e. enamel formation, U.S. Pat. No. 4,672,032 (Slavkin)),binding between hard tissues (EP-B-0 337 967 and EP-B0 263 086) and openwound healing, such as of skin and mucosa (WO 9943344).

The present application relates to the beneficial effects of EMD onguided connective soft tissue growth and resistance to contraction inclosed wounds following loss or removal of significant volumes oftissue, such as e.g. after tumour surgery and especially in combinationwith radiation therapy, effects that are both unexpected and surprising.

DISCLOSURE OF THE INVENTION

Maintenance, repair and regeneration of differentiated tissue is guidedby several tissue specific growth factors. However, so far all attemptsto apply individual growth factors to regeneration of human tissue intherapy have failed in clinical trials. This is thought to be due togrowth factors being pluripotent signal factors that work in concert. Inan intricate teamwork, they induce and modulate tissue growth,differentiation and maturation during development and orchestratehealing, repair and regeneration of diseased tissue. The enamel matrixderivatives (EMD) of the present invention, however, are able tocircumvent this problem, as they can induce not only one but anorchestrated cascade of factors naturally found in tissues developingadjacent to the enamel matrix. They mimic the natural environment of adeveloping tissue and thus mimic a natural stimulation for tissueregeneration, cell differentiation and/or maturation.

The present invention is based on the surprising finding that enamelmatrix, enamel matrix derivatives and/or enamel matrix proteins (theterm “an active enamel substance” is in the following also used for anenamel matrix, an enamel matrix derivative or an enamel matrix protein)do not only promote periodontal ligament cell growth, but also stimulatenon-periodontal fibroblast cell growth and differentiation, whereasepithelial cell growth or differentiation is not stimulated by thepresence of active enamel substances. The use of a pharmaceutical orcosmetical composition comprising an active enamel substance, asdescribed in the present invention, thus relates to the selectivestimulation of mesodermal and/or endodermal cell growth, includinggrowth of cartilage, bone and connective tissue, striated and smoothmuscles, the heart, blood and lymph vessels and cells, the kidneys,gonades (ovaries and testes), the genital ducts, serous membranes liningthe body cavities (pericardial, pleural and peritoneal), the spleen, thecortex of the suprarenal gland, the epithelial lining of thegastrointestinal and respiratory tracts, the parenchyma of the tonsils,thyroid gland, parathyroid glands, thymus, liver and pancreas, theepithelial lining of the urinary bladder and most of the ureta, theepithelial lining of the tympanic cavity, tympanic antrum and auditorytube, as opposed to ectodermal cell growth including growth of thecentral or peripheral nervous system, the epidermis and its appendages(hair and nails), the mammary glands, pituitary gland, the subcutaneousglands and the enamel of teeth, which is not stimulated by theapplication of said composition comprising an active enamel substance.

As shown by the present inventors (see experiment 1), the increasedattachment rate of non-periodontal fibroblast cells that grow on activeenamel substances demonstrates that an enamel protein based matrixmimics an extracellular matrix. This mimicry facilitates rapidattachment of these cells. The observed rise in growth rate andmetabolism in these fibroblast cells, growing on active enamelsubstances, further proves that active enamel substances provide anextracellular matrix that stimulates fibroblast cells to speed up theirmetabolism. Also, a rise in growth rate is reflected in the increase ofDNA synthesis, indicating that cell proliferation is up-regulated inthese cultures. Furthermore, since the increase in utilisation of[³⁵S]-methionine in these fibroblast cells exceeds the rise in growthrate, some of the added metabolic activity also reflects a boostedanabolism and/or secretion of extracellular proteins.

The surprising findings described above lead to the envisioning ofsubstantially new possibilities for the use of active enamel substances.One embodiment, described in the present invention, comprises the use ofactive enamel substances for preparing a pharmaceutical or cosmeticalcomposition that is to be used as a fill-in application for significanttissue loss wounds that would otherwise lead to painful and/ordisfiguring scarring of a mammal's body. The successful application ofsaid composition, as described herein, leads to increased neogenesis andtissue-specific gain of soft tissue, muscle, blood and lymph vessels,tendons, and cartilage.

Not intended to be within the scope of the present invention is thetreatment of shallow open wounds in soft tissue with active enamelsubstances.

A preferred embodiment of the present invention relates to the use of anactive enamel substance for the preparation of a pharmaceutical orcosmetic composition that is used as a beneficial agent for theenhancement or improvement of guided connective tissue growth into softtissue defects following significant tissue loss due to trauma,infectious diseases, necrosis, removal of neoplasms or othercytoreductive surgical interventions. As demonstrated in theexperimental section herein, the active enamel substance exertsespecially useful effects in guiding and stimulating connective tissuegrowth into a significant tissue loss following the surgical removal ofa tumour. Further, another preferred embodiment relates to the use ofsaid active enamel substance for the preparation of a pharmaceutical orcosmetic composition for guiding and stimulating connective tissuegrowth following post surgical tumour treatment after significant tissueloss, such as after radiation therapy.

The present invention thus provides optimal means to refill a loss oftissue that is left from a surgically removed tissue, such as e.g. atumour, with new fibroblasts that are stimulated to proliferate in thewound cavity. To this means, the cavity is filled with a preparation ofactive enamel substances, such as e.g. with EMDOGAIN® (BIORA AB,Sweden), which will successively be degraded and replaced by newfibroblasts. Thereby, the cavity will be protected from collapsing andthe surrounding tissue will be protected from contracting, and finally,the new grown soft tissue will act as a natural replacement for thesurgically removed tissue. Furthermore, the unique property of activeenamel substances to stimulate proliferation and differentiation ofselective tissue types, will in the above mentioned embodiment onlystimulate the neogenesis of mesodermal or endodermal cells, but notstimulate any ectodermal cell growth.

In an especially preferred embodiment of the invention, an active enamelsubstances can therefore be used for the preparation of a pharmaceuticalor cosmetic composition for filling of a cavity wound that ischaracterised by substantial loss of tissue after surgical removal of abreast tumour. Breast tumours are mainly glandular tumours, i.e. thatactive enamel substances will not stimulate the regrowth of the tumourtissue, glands being of ectodermal origin, but only stimulate thefill-in of the cavity with new connective tissue. In a later scenario, aperson skilled in the art will have no difficulties to further envisionthe stimulation of regrowth of blood vessels and innervation that willfollow the fibrotic fill-in of the wound.

The use of a composition comprising an active enamel substance, asdescribed in the present invention, will prevent contraction ofconnective tissue, following significant tissue loss due to surgicaltumour treatment such as surgical removal of significant tumour tissueand post surgical tumour treatment, such as, for example but notexclusively, radiation therapy. The use of active enamel substances inaccordance to this invention in a tension-free closure may be associatedwith less pain and less incidence of postoperative fluid accumulation(seroma). Furthermore, compositions comprising an active enamelsubstance will also help increase tensile strength of the wound and thusbe beneficial for improving yet another aspect of the repair process.

Accordingly, the invention relates to the use of an active enamelsubstance for the preparation of a pharmaceutical or cosmeticcomposition i) for improving connective tissue fill of a significanttissue loss and/or defect, and/or ii) for avoiding soft tissuecontraction following significant tissue loss due to e.g. tumour removaland thus limiting post surgical therapy complications.

LEGENDS TO FIGURES

FIG. 1: Normal Human Dermal Fibroblast (NHDF) cell attachment rateduring the first hours after seeding is nearly five times more efficientwhen the surface of the culture dish is coated with EMD. n=6 error barsgive ±SD

FIG. 2: Density plot of cultured NHDF cells show that cells growing inpresence of EMD proliferate faster than controls. Five different areasin each culture were counted in each of six parallel cultures at eachtime point (n=30), error bars are ±SD.

FIG. 3: Utilization of [³⁵S]methionine pulse plotted against days inNHDF cultures after seeding. The presence of EMD in the culturessignificantly increases the metabolic rate of NHDF cells. n=9, errorbars give ±SD.

FIG. 4: DNA synthesis analyzed by BrdU incorporation during DNAreplication show that NHDF cells significantly increase synthesis ofnucleic acids in presence of EMD. n=6, error bars give ±SD.

FIG. 5: Radiated cells growing in the presence of EMD increase theirnumber twice as fast as unstimulated cultures (FIGS. 5A and 5B)

DETAILED DISCLOSURE

The present invention relates to the use of enamel matrix, enamel matrixderivatives and/or enamel matrix proteins for actively inducing, guidingand/or stimulating connective tissue growth and for being involved inpreventing connective tissue scaring and/or contraction in deep cavitywound healing.

The majority of tissue cavities and defects are filled throughreparative processes, i.e. the new tissue that is formed (scar tissue)is often of different volume and structurally and chemically unlike theoriginal tissue. In the early stage of the tissue repair, one process isalmost always involved, the formation of a transient connective tissuein the area of the tissue injury. This process starts by forming of anew extracellular collagen matrix by fibroblasts. Said new extracellularcollagen matrix supports the connective tissue during the final healingprocess. However, in defects wherein a significant volume of tissue hasbeen removed or lost, the defect or deep tissue-cavity will not fillwith connective tissue and extracellular collagen based matrix, but asignificant part of the defect volume will fill with cell free exudatefrom the surrounding tissue. To allow for connective tissue fill of thewhole cavity, the exudate has to be drained out so that matrix formationand subsequent connective tissue growth can occupy most of the cavity.

Under normal circumstances, the body provides mechanisms for healingtissue cavities in order to restore the function and integrity of theinvolved tissue or body part. However, the recovering time can be verylong and the defect may persist for an extended period of time, i.e.months or even years. During this time, the patient is often disabledand suffers from pain, discomfort and complications that need regularprofessional attendance.

The repair of tissue defects and/or cavities follows the classical woundhealing stages that normally include inflammation (normally 1–3 days),migration (normally 1–6 days), proliferation (normally 3–24 days) andmaturation (normally 1–12 months). The healing process is a complex andwell-orchestrated physiological process that involves migration,proliferation and differentiation of a variety of cell types as well assynthesis of matrix components.

The healing process may be separated into the following three phases:

i) Haemostasis and Inflammation

When platelets are present outside the circulatory system and exposed tothrombin and collagen, they become activated and they aggregate. Thus,platelets initiate the repair process by aggregating and forming atemporary plug to ensure haemostasis and prevent invasion from bacteria.The activated platelets initiate the coagulation system and releasegrowth factors like platelet-derived growth factor (PDGF) and epidermalgrowth factors (EGFs) and transforming growth factors (TGFs).

The first cells to invade the wound area are neutrophils followed bymonocytes which are activated by macrophages.

The major role of neutrophils appears to be clearing the wound ofcontaminating bacteria or defending the wound against contaminatingbacteria and to improve the healing of the wound by removing dead cellsand platelets. The infiltration of neutrophils ceases within about thefirst 48 hours, provided that no bacterial contamination is present inthe wound. Excess neutrophils are phagocytosed by tissue macrophagesrecruited from the circulating pool of blood-borne monocytes.Macrophages are believed to be essential for efficient wound healing inthat they are also responsible for phagocytosis of pathogenic organismsand a clearing up of tissue debris. Furthermore, they release numerousfactors involved in subsequent events of the healing process. Themacrophages attract fibroblasts that start the production of collagen.

ii) Granulation Tissue Formation

Within 48 hours after wounding, fibroblasts begin to proliferate andmigrate into the wound space from the connective tissue at the woundedge. The fibroblasts produce collagens and glycosaminoglycans and interalia low oxygen tension at the wound stimulates proliferation ofendothelial cells. The endothelial cells give rise to the formation of anew capillary network. At this stage, the wound area, i.e. the cavity,is further decreased by contraction. In the case of deep, cavity-like,soft tissue enclosed defects, e.g. like those present following surgicalremoval of a breast tumour, this contraction affects both the appearanceof the body part as well as the performance, and often causes pain anddiscomfort.

At this stage, if necessary as adjuvant therapy after removal ofmalignant tumours, radiation therapy is applied. Said therapy aims atremoving residual cancer cells and local metastases from the tissue orbody part that has been treated. Besides killing all proliferatingcancer cells, this therapy also affects all normal cells that areundergoing mitosis, and imposes structural damage to DNA and proteins inthe repairing tissue. The effect of this damage is a severe slow down ofthe cellular repair processes, while the extracellular processes remainmostly unaffected. With time, this induced imbalance between cellulargrowth and extracellular processes causes severe contractions of thewound surfaces and thus the cavity and/or defect. These contractions areoften so painful and disfiguring that the tissue or body part inquestion is removed and substituted by prosthesis where possible, e.g.removal of major part of the breast and reconstruction by silicon inlay.In the cases where removal is not compatible with life, reconstructivesurgery is the only option. However, because of the radiation damage tothe DNA, the tissue does not have the ability to conduct the normalrepair sequence and the chances are that the new surgical defect alsoundergoes contraction with subsequent need for even more reconstructivesurgery.

iii) Tissue Remodelling

As soon as the defect is completely filled with scar tissue, remodellingof the tissue begins. During this phase the scar tissue is substitutedwith a more organised type of tissue that aims at restoring thestrength, function, performance and appearance of the tissue/body partin question. This phase typically lasts for several years after theinitial defect. Also this stage is severely hampered by radiationtherapy that causes contraction and scarring.

All of the above-mentioned processes take considerable time. The rate ofhealing is influenced by the wound's freedom from infection, the generalhealth of the individual, presence of foreign bodies, etc. Somepathologic conditions like infection, maceration, drying out, generallypoor health and malnutrition can, if left untreated for a longer time,lead to formation of a chronically inflamed or infected tissue defect orcavity that is very difficult to cure. Furthermore, since the primarydefect is caused by removal of significant volumes of tissue, surgicalremoval of the defunct tissue is not desirable or even possible. Theseconditions therefore severely affect the quality of life of a patientand can also be extremely disabling or even life threatening.

Traditionally, cavity like, soft tissue defects have been treated byclosing them with sutures with a draining device present that allowswound exudate and puss to escape from the cavity during the initialphases of the healing process. While favourable for cellular filling ofthe defect, this strategy delays the healing because a foreign body isleft in the wound that can provoke inflammation and makes a gateway forinvading micro flora. Delayed wound healing or inflammation canexacerbate fibrosis and subsequent tissue contraction.

Until the tissue defect is filled with cellular connective tissue, thedefect remains at risk of continued or new infection, inflammationand/or severe contraction. Therefore, the quicker the defect can heal,the sooner the risk is removed. Thus, the use of active enamelsubstances according to the present invention represent means that caninfluence the rate of connective tissue filling and organization andfavorably influence the healing capacity of radiated and/or non-radiatedconnective tissues. Furthermore, as almost all tissue repair processesinclude the early connective tissue formation, a stimulation of this andthe subsequent processes are also contemplated to improve the qualityand quantity of tissue defect filling.

In the present context, the term “clinical healing” is used to denote asituation wherein no tissue interruption can be visually observed andonly discrete signs of inflammation are present, such as a light rednessor a discretely swollen tissue. In addition, no complaints of pain arepresent when the organ is relaxed or untouched.

As mentioned above, the invention relates to the use of enamel matrix,enamel matrix derivatives and/or enamel matrix proteins as an agent forstimulation of connective tissue growth, i.e. as an agent thataccelerates, stimulates and/or promotes the growth of connective tissuecells. Accordingly, an important aspect is the use of an active enamelsubstance as a repair agent to prevent wound contraction, both surgicaland radiation induced. Furthermore, secondarily to the biologicaleffect, the administration of active enamel substances will render painrelief, because of a more rapid filling process of the defect and/ortissue-cavity and less wound contraction.

The enamel matrix, enamel matrix derivatives and/or enamel matrixproteins may be applied either directly into the soft tissue defectprior to suturing or it may be injected into the wound cavity aftersuturing. The volume/amount of enamel matrix, enamel matrix derivativesand/or enamel matrix proteins applied will differ from case to case andtissue to tissue, but generally, the therapy will aim at replacing thevolume of the lost tissue. However, in cases where an increase ordecrease of the volume of a tissue or body part, e.g. a breast, isdesirable, the enamel matrix, enamel matrix derivatives and/or enamelmatrix proteins may be applied in surplus or deficit to acquire thedesired outcome. The active enamel substance may be used as such, or maybe used in a suitable preparation or pharmaceutical composition.

The present inventors have in experiments with cultured dermalfibroblasts shown that cells that have received therapeutic dosages ofionising radiation can be stimulated with EMDOGAIN® (BIORA AB, Sweden)to replicate and grow almost at the speed of normal untreated cells. Theability of radiated cells to recover from the damage and to restorenormal functions is crucial if contraction complications are to beavoided following cancer surgery and radiation therapy. Thus, theobservation that active enamel substances can induce such beneficialchanges in radiated fibroblasts proves the concept of the presentinvention.

Moreover, the inventors have found that active enamel substances havethe ability to stimulate fibroblast invasion, proliferation and growth.Furthermore, there are indications that the application of active enamelsubstances leads to improved defect fill that reduces post operation andpost radiation contractions in the defect. Also, the inventors haveobserved that the inflammation stage is shortened and the typical signssuch as warmth, redness, oedema and pain are less noticeable, and thatnew tissue is formed more rapidly after application of active enamelsubstances. Thus, the observed time for wound healing (e.g. aftersurgery) is significantly shortened as compared to surgery without theuse of active enamel substances.

Interestingly, the inventors have also observed that various cellcultures of fibroblasts (embryonic, dermal, derived from the periodontalligament, fish fin or bird skin), produce twice as much transforminggrowth factor (TGF-β1) when stimulated with EMDOGAIN® (BIORA AB,SWEDEN), compared to non-stimulated cultures. As TGF-β1 is considered tobe of central importance in the neogenesis and reorganization ofconnective tissue, these findings further support the concept of thepresent invention.

Significant tissue loss or defect following trauma or cytoreductivesurgery normally produces cavities at the location of the removedtissue. These cavities usually fill with fluid and/or fibrous, densescar tissue, causing the function and appearance of the remaining tissueto be severely impaired. If normal connective tissue growth is inducedto fill such cavities, it dramatically improves the functional andesthetical outcome of cytoreductive surgery and trauma treatment.

The preparation of an active enamel substance according to the presentinvention is effective for treating a wide variety of different typesand sizes of wounds. Regeneration of experimentally provoked periodontalwounds have previously been described by the inventors and is notintended to be within the scope of the present invention, neither ishealing of wounds in skin or mucous membranes.

In the present context the terms “wound cavity”, “tissue cavity” and“tissue defects” denote an internal bodily injury with disruption of thenormal integrity of tissue structures that follows from a procedureand/or trauma that involves removal and/or loss of a significant amountof tissue from the body or a body part. The term is also intended toencompass the terms “surgical lesions”, “necrosis”, “abscesses”,“mucocele”, “cysts”, “fistulas”, “excavations”, “alveola” and all otherterms used to describe abnormal cavities within the human body ortissue.

A deep cavity-shaped wound is in the present context clearly defined incontrast to a shallow, open wound on the surface of a tissue, especiallyon the epidermis of a body, such as an abrasive-wound or a mucosalwound. A cavity-shaped wound creates a deep, substantially hollow spacein the injured tissue and will either fill with fluids, scar tissueand/or collapse in the natural repair process.

Another way of classifying wound cavities or tissue defects is as i)significant tissue loss due to surgical incisions, radiation treatment,abrasions, lacerations, burns (chemical and thermal), donor site woundsand substantial bites, or as ii) significant tissue loss due topathological conditions including ischemic ulcers, fistulae, neoplasms,tumours, hammartomas, infections, necrosis, and infarctions.

A “significant tissue loss” in the context of the present invention ismeant to comprise an amount of tissue that is typically removed from anoriginal tissue, for example but not exclusively, due to trauma or dueto surgery, and that results in a deep wound in said tissue and/or thatimpairs function and/or appearance of said body part. A “significanttissue loss” in the context of the present invention will ultimatelylead to scarring.

In one embodiment of the invention, significant tissue loss comprisese.g. the loss of at least 5% of weight of tissue, compared to theoriginal weight of an organ or body part. Such a loss will in thiscontext comprise a reduction of tissue mass of between 5% and 75%, suchas between at least 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or at least between 20%and 75%. In another embodiment of the present invention, the tissue losscomprises a reduction of tissue weight of at least 25%, such as at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or at least 85%.

In an especially preferred embodiment of this invention, the use of anactive enamel substance for the preparation of a pharmaceutical orcosmetic composition for filling a tissue cavity and/or defect that ischaracterised by a substantial tissue loss is comprised, wherein thetissue loss is due to the surgical removal of a tumour and/or postsurgical treatment with radiation therapy. In this specific embodiment,“significant tissue loss” in the context of the present invention ismeant to comprise an amount of tissue ranging between about 1 ml–250 ml.Such a range will thus include an amount of tissue of about 1 ml, 1.25ml, 1.5 ml, 2 ml, 2.25 ml, 2.5 ml, 2.75 ml, 3 ml, 3.25 ml, 3.5 ml, 3.75ml, 4 ml, 4.25 ml, 4.5 ml, 4.75 ml, 5 ml, 5.25 ml, 5.5 ml, 5.75 ml, 6ml, 6.25 ml, 6.5 ml, 6.75 ml, 7 ml, 7.25 ml, 7.5 ml, 7.75 ml, 8 ml, 8.25ml, 8.5 ml, 8.75 ml, 9 ml, 9.25 ml, 9.5 ml, 9.75 ml, 10 ml, 15 ml, 20ml, 25 ml, 50 ml, 75 ml, 100 ml, 150 ml, 200 ml or 250 ml.

In certain embodiments, wherein the tissue loss is e.g. due to thesurgical removal of the majority of an organ or body part and/or a largetumour, the amount of tissue will of course include even larger volumesthan 250 ml, such as between at least 250 ml and 500 ml, depending onthe original size of the organ or body part and/or tumour.

The kinds of tissue cavities to be treated according to the inventioninclude diverse orbital cavity or periorbital soft tissue and bonydefects due to trauma, removal of benign or malignant neoplasms, tumoursof the head and neck, abdomen and/or the extremities, particularlyovarian cancer and/or prostate cancer. They further comprise closedabdominal wounds, cavity wounds with negative pressure, penetratingthoracic and abdominal trauma wounds and/or abdominal gunshot injuries.

In the present context, a “tumour” stands for any new-growth of tissuein which the multiplication of cells is uncontrolled and progressive.The term “tumour” is herein equivalent to “neoplasm”.

The invention comprises the use of an active enamel substance for thepreparation of a pharmaceutical or cosmetic composition for filling awound cavity and/or tissue defect that is due to surgical removal ofprimary and/or metastatic solid tumors and carcinomas of the breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder, bile duct, small intestine, urinary tractincluding kidney, bladder and urothelium, female genital tract includingcervix, uterus, ovaries, choriocarcinoma and gestational trophoblasticdisease, male genital tract including prostate, seminal vesicles, testesand germ cell tumors, endocrine glands including thyroid, adrenal, andpituitary, skin including hemangiomas, melanomas, sarcomas arising frombone or soft tissues and Kaposi's sarcoma, tumors of the head, nerves,eyes, and meninges including astsrocytomas, gliomas, glioblastomas,retinoblastomas, neuromas, neuroblastomas, Schwannomas and meningiomas,solid tumors arising from hematopoietic malignancies such as leukemiasand including chloromas, plasmacytomas, placques and tumors of mycosisfungoides, and cutaneous T-cell lymphoma/leukemia and/or lymphomasincluding both Hodgkin's and non-Hodgkin's lymphoma's.

In one embodiment of the present invention, tissue removed by resectionduring surgery includes not only tissue suspected by the surgeon ofbeing neoplastic, but also includes an amount of healthy tissue takenbecause the precise tumour margins can not be ascertained by thesurgeon. Coupled with the devastating risk of not removing neoplastictissue resulting in tumour recurrence, surgical protocol dictates thathealthy tissue is taken in order to ensure the removal of neoplastictissue. Of course, the final determination as to whether the resectedtissue is malignant falls to the pathologist who receives the tissueremoved during the surgical procedure.

The present invention further relates to the use of an active enamelsubstance for the preparation of a pharmaceutical composition for areconstruction that can be a post-mastectomy procedure, a post-traumaticprocedure, or a procedure done to enlarge or decrease the volume of abreast. A reconstruction can be contemporaneous with a mastectomy or canbe delayed, taking place over one or more post-mastectomy surgicalprocedures. In accordance with the invention, a delayed procedurecomprises: a multistage procedure where a mastectomy is performed withcontemporaneous placement of an expander, and a subsequent procedurewhen the reconstruction is performed; a mastectomy; a subsequentprocedure when an expander is placed, and a subsequent procedure whenreconstruction is performed; revisions to a previous reconstruction; or,the placing or modifying of breast implant materials that comprise apharmaceutical composition according to the present invention.

Thus, the invention provides means for total and/or partial breastreconstruction that is either delayed or immediate. With an immediatereconstruction, the patient does not experience a mastectomy deformityand accompanying emotional trauma; however, for many women a delayedreconstruction is medically indicated. For patients who have undergone astandard modified radical mastectomy, delayed autologous reconstructionis accomplished after expansion of the skin envelope.

By use of preferred embodiments, a breast reconstruction according tothe present invention can be performed on both breasts. Thus, if asubsequent breast cancer occurs in the contralateral breast, the sameprocedure can be performed. Alternatively, if a bilateral breast canceris present, one or both breasts can be reconstructed with thistechnique.

As used herein, a “substantially circumareolar incision” comprises anincision that circumscribes at the perimeter of the nipple-areolarcomplex; in instances where a breast reduction and/or a nipple-areolarrepositioning is to be performed, an incision that circumscribes at theperimeter of the nipple-areolar complex and includes additional breastskin; and, an incision that approximates the areolar perimeter yet iswithin the area of the areola. Preferably, particularly for immediateembodiments of the invention, the circumareolar incision closelycorresponds to the perimeter of the areola, and is at or within themargin of the areola. A substantially circumareolar incision alsocomprises radial or wedge skin incisions at the border of the areola.Radial or wedge skin incisions at the border of the areola are lessideal since they create scarring that does not correspond to a naturaltissue plane.

Advantageously, a reconstructed breast produced in accordance with thepresent invention has better contour and projection than reconstructedbreasts that resulted from procedures well known to the skilled artisan.

Preferred embodiments of the procedure of the present invention areperformed following a circumareolar mastectomy. A circumareolarmastectomy eliminates the transverse mastectomy scar that was aconsequence of prior reconstruction procedures. To make mostadvantageous use of a circumareolar mastectomy, it is preferred that thesurgeon limit any biopsy and subsequent mastectomy skin excision to theregion of the nipple-areolar complex. When the skin excision in amastectomy is limited to the region of the nipple-areolar complex, theskin envelope of the breast is completely preserved. Since the biopsyincision is generally removed at the time of the mastectomy, biopsyincisions outside the region of the areola often necessitate anon-preferably large skin incision on the breast skin envelope. With alarge skin incision, the mastectomy scar is not camouflaged at theborder of the areola, and the reconstructed breast is less likely tohave a normal contour.

A mastectomy excision through a substantially circumareolar incisionthat is larger than the areolar perimeter can be performed in accordancewith the invention, particularly for patients for whom a breastreduction and/or nipple-areolar repositioning is indicated; for suchpatients, a “substantially circumareolar incision” comprises an incisionthat corresponds to the perimeter of the areola and comprises anincision that corresponds to a standard pattern reduction orrepositioning incision. In general, if a skin resection extends beyondthe perimeter of the nipple-areolar complex, the resulting mastectomyscar is more readily apparent.

Alternatively, yet also in accordance with the present invention, avariable amount of areola may be left with the breast skin.

Radiation therapy is known to those skilled in the art to be lessdistorting if performed after a breast reconstruction. Patientsnecessitating postoperative radiation therapy will be candidates for theuse of the present invention. The use will alter the patient'spost-operative prognosis favourably. Size or stage of the breast cancerwill not limit applicability of the procedure. If a modified radicalmastectomy with a large elliptical skin excision is indicated, a delayedreconstruction in accordance with the invention is generally used.

Additionally to the use in breast surgery, the present invention furtherrelates to the use of an active enamel substance for the preparation ofa pharmaceutical composition for reconstruction or for guided connectivetissue growth after vaginal, urinal and/or anal surgery or surgery onurine bladder, womb or intestines.

In the scope of the present invention are also other uses for repairingsoft tissue defects, such as soft tissue defects resulting fromincisional hernias and soft tissue defects resulting from extirpativetumour surgery. Other applications of the present invention includelaparoscopic inguinal hernia repair, standard inguinal hernia repair,umbilical hernia repair, paracolostomy hernia repair, femora herniarepair, lumbar hernia repair, and the repair of other abdominal walldefects, thoracic wall defects and diaphragmatic hernias and defects.

In yet another embodiment, the present invention relates to the use ofan active enamel substance for the preparation of a pharmaceutical orcosmetic composition for the enlargement of a breast or any other softtissue of a mammal, wherein a certain amount of said composition, isinserted into a mammalian tissue without any prior loss of tissue. Thisembodiment incorporates the use of an active enamel substance for purelycosmetic reasons and is not limited to a post-surgical treatment of atumour, such as e.g. breast cancer. In this context, the scope of theinvention therefore relates to cosmetic methods for treating a humanbeing with an active enamel substance for stimulating the neogenesis ofsoft tissue, characterised by inserting or injecting a suitablepharmaceutical composition comprising active enamel substances, such ase.g. EMDOGAIN® (BIORA AB, Sweden) into the organ and/or body part,wherein cosmetical enlargement or filling is desired.

Enamel matrix is a precursor to enamel and may be obtained from anyrelevant natural source, i.e. a mammal in which teeth are underdevelopment. A suitable source is developing teeth from slaughteredanimals such as, e.g., calves, pigs or lambs. Another source is e.g.fish skin.

Enamel matrix can be prepared from developing teeth as describedpreviously (EP-B-0 337 967 and EP-B-0 263 086). The enamel matrix isscraped off and enamel matrix derivatives are prepared, e.g. byextraction with aqueous solution such as a buffer, a dilute acid or baseor a water/solvent mixture, followed by size exclusion, desalting orother purification steps, followed by freeze-drying. Enzymes mayalternatively be deactivated by treatment with heat or solvents, inwhich case the derivatives may be stored in liquid form withoutfreeze-drying.

As an alternative source of the enamel matrix derivatives or proteinsone may also use generally applicable synthetic routes, well known to aperson skilled in the art, or use cultivated eukaryotic and/orprokaryotic cells modified by DNA-techniques. The enamel matrix proteinsmay thus be of recombinant origin and alternatively genetically modified(see, e.g., Sambrook, J. et al.: Molecular Cloning, Cold Spring HarborLaboratory Press, 1989).

In the present context, enamel matrix derivatives are derivatives ofenamel matrix which include one or several enamel matrix proteins orparts of such proteins, produced naturally by alternate splicing orprocessing, or by either enzymatic or chemical cleavage of a naturallength protein, or by synthesis of polypeptides in vitro or in vivo(recombinant DNA methods or cultivation of diploid cells). Enamel matrixprotein derivatives also include enamel matrix related polypeptides orproteins. The polypeptides or proteins may be bound to a suitablebiodegradable carrier molecule, such as polyamine acids orpolysaccharides, or combinations thereof. Furthermore, the term enamelmatrix derivatives also encompass synthetic analogous substances.

Proteins are biological macromolecules constituted by amino acidresidues linked together by peptide bonds. Proteins, as linear polymersof amino acids, are also called polypeptides. Typically, proteins have50–800 amino acid residues and hence have molecular weights in the rangeof from about 6,000 to about several hundred thousand Dalton or more.Small proteins are called peptides or oligopeptides.

Enamel matrix proteins are proteins that normally are present in enamelmatrix, i.e. the precursor for enamel (Ten Cate: Oral Histology, 1994;Robinson: Eur. J. Oral Science, January 1998, 106 Suppl. 1:282–91), orproteins which can be obtained by cleavage of such proteins. In general,such proteins have a molecular weight below 120,000 Dalton and includeamelogenins, non-amelogenins, proline-rich non-amelogenins andtuftelins.

Examples of proteins for use according to the invention are amelogenins,proline-rich non-amelogenins, tuftelin, tuft proteins, serum proteins,salivary proteins, ameloblastin, sheathlin, and derivatives thereof, andmixtures thereof. A preparation containing an active enamel substancefor use according to the invention may also contain at least two of theaforementioned proteinaceous substances. Moreover, other proteins foruse according to the invention are found in the marketed productEMDOGAIN® (BIORA AB, Sweden).

EMDOGAIN® (BIORA AB, S-205 12 Malmö, Sweden) contains 30 mg EnamelMatrix protein, heated for 3 hours at about 80° C. in order toinactivate residual proteases, and 1 ml Vehicle Solution (PropyleneGlycol Alginate), which are mixed prior to application, unless theprotein and the Vehicle are tested separately. The weight ratio is about80/8/12 between the main protein peaks at 20, 14 and 5 kDa,respectively.

In general, the major proteins of an enamel matrix are known asamelogenins. They constitute about 90% w/w of the matrix proteins. Theremaining 10% w/w includes proline-rich non-amelogenins, tuftelin, tuftproteins, serum proteins and at least one salivary protein; however,other proteins may also be present such as, e.g., amelin (ameloblastin,sheathlin) which have been identified in association with enamel matrix.Furthermore, the various proteins may be synthesised and/or processed inseveral different sizes (i.e. different molecular weights). Thus, thedominating proteins in enamel matrix, amelogenins, have been found toexist in several different sizes that together form supramolecularaggregates. They are markedly hydrophobic substances that underphysiologically conditions form aggregates. They may carry or becarriers for other proteins or peptides.

Other protein substances are also contemplated to be suitable for useaccording to the present invention. Examples include proteins such asproline-rich proteins and polyproline. Other examples of substances thatare contemplated to be suitable for use according to the presentinvention are aggregates of such proteins, of enamel matrix derivativesand/or of enamel matrix proteins as well as metabolites of enamelmatrix, enamel matrix derivatives and enamel matrix proteins. Themetabolites may be of any size ranging from the size of proteins to thatof short peptides.

As mentioned above, the proteins, polypeptides or peptides for useaccording to the invention typically have a molecular weight of at themost about 120 kDa such as, e.g., at the most 100 kDa, 90 kDa, 80 kDa,70 kDa or 60 kDa as determined by SDS PAGE electrophoresis.

The proteins for use according to the invention are normally presentedin the form of a preparation, wherein the protein content of the activeenamel substance in the preparation is in a range of from about 0.05%w/w to 100% w/w such as, e.g., about 5–99% w/w, about 10–95% w/w, about15–90% w/w, about 20–90% w/w, about 30–90% w/w, about 40–85% w/w, about50–80% w/w, about 60–70% w/w, about 70–90% w/w, or about 80–90% w/w.

A preparation of an active enamel substance for use according to theinvention may also contain a mixture of active enamel substances withdifferent molecular weights.

The proteins of an enamel matrix can be divided into a high molecularweight part and a low molecular weight part, and it has been found thata well-defined fraction of enamel matrix proteins possesses valuableproperties with respect to treatment of periodontal defects (i.e.periodontal wounds). This fraction contains acetic acid extractableproteins generally referred to as amelogenins and constitutes the lowmolecular weight part of an enamel matrix (cf. EP-B-0 337 967 and EP-B-0263 086).

The low molecular weight part of an enamel matrix has a suitableactivity for inducing binding between hard tissues in periodontaldefects. In the present context, however, the active proteins are notrestricted to the low molecular weight part of an enamel matrix. Atpresent, preferred proteins include enamel matrix proteins such asamelogenin, tuftelin, etc. with molecular weights (as measured in vitrowith SDS-PAGE) below about 60,000 Dalton but proteins having a molecularweight above 60,000 Dalton have also promising properties as candidatesfor promoting connective tissue growth.

Accordingly, it is contemplated that the active enamel substance for useaccording to the invention has a molecular weight of up to about 40,000such as, e.g. a molecular weight of between about 5,000 and about25,000.

By separating the proteins, e.g. by precipitation, ion-exchangechromatography, preparative electrophoresis, gel permeationchromatography, reversed phase chromatography or affinitychromatography, the different molecular weight amelogenins can bepurified.

The combination of molecular weight amelogenins may be varied, from adominating 20 kDa compound to an aggregate of amelogenins with manydifferent molecular weights between 40 and 5 kDa, and to a dominating 5kDa compound. Other enamel matrix proteins such as tuftelin orproteolytic enzymes normally found in enamel matrix can be added andcarried by the amelogenin aggregate.

In general, the enamel matrix, enamel matrix derivatives and enamelmatrix proteins are hydrophobic substances, i.e. less soluble in water,especially at increased temperatures. In general, these proteins aresoluble at non-physiological pH values and at a low temperature such asabout 4–20° C., while they will aggregate and precipitate at bodytemperature (35–37° C.) and neutral pH.

The enamel matrix, enamel matrix derivatives and/or enamel matrixproteins for use according to the invention comprise an active enamelsubstance, wherein at least a part is in the form of aggregates or afterapplication in vivo is capable of forming aggregates. The particle sizeof the aggregates is in a range of from about 20 nm to about 1 μm.

In accordance to the present invention, an active enamel substance maybe used together with other active drug substances such as, e.g.anti-bacterial, anti-inflammatory, antiviral, antifungal substances orin combination with local chemotherapy, inducers of apoptosis, growthfactors such as, e.g., TGFβ, PDGF, IGF, FGF, EGF, keratinocyte growthfactor or peptide analogues thereof. Enzymes—either inherently presentin the enamel matrix or preparation thereof or added—may also be used incombination with an enamel matrix, enamel matrix derivative and/orenamel matrix protein, especially proteases.

A preparation of an active enamel substance is normally formulated as apharmaceutical or cosmetic composition. Such a composition may of courseconsist of the proteinaceous preparation or it may further comprise apharmaceutically or cosmetically acceptable excipient. Especiallysuitable excipients for use in pharmaceutic or cosmetic compositions arepropylene glycol alginate, or hyaluronic acid or salts or derivativesthereof.

In the following, examples of suitable compositions containing an activeenamel substance(s) are given. Depending on the use of the active enamelsubstance(s), a composition may be a pharmaceutical or a cosmeticcomposition. In the following the term “pharmaceutical composition” isalso intended to embrace cosmetic compositions as well as compositionsbelonging to the so-called grey area between pharmaceuticals andcosmetics, namely cosmeceuticals.

For the administration to an individual (an animal or a human), anactive enamel substances and/or a preparation thereof is preferablyformulated into a pharmaceutical composition containing the activeenamel substance and, optionally, one or more pharmaceuticallyacceptable excipients.

A composition comprising the active enamel substance to be administeredmay be adapted for administration by any suitable route, e.g. by topicaladministration through a syringe, or by administration to a tissuecavity through a hose or draining device. Furthermore, a composition maybe adapted to administration in connection with surgery, e.g. inconnection with incision within the body in order to promote repair andfilling of deep cavity-shaped wounds, substantial tissue losses aftersurgery and tissue defects.

As mentioned above, a composition of the active enamel substance(s) maybe suitable for use during surgery, e.g. for local application (e.g. ina breast or in the abdominal wall) in the form of a gel, film or drypellet to induce fibroblast invasion, proliferation and growth.

The compositions may be formulated according to conventionalpharmaceutical practice, see, e.g., “Remington's PharmaceuticalSciences” and “Encyclopedia of Pharmaceutical Technology”, edited bySwarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988.

As mentioned above, the application of a composition comprising anactive enamel substance is intended for inducing and stimulatingconnective tissue cell invasion, proliferation and growth as part of arepair process following significant removal or loss of tissue. Otherapplications may of course also be relevant such as application directlyin or into a deep wound or other substantial tissue defects.

A pharmaceutical composition comprising an active enamel substanceserves as a drug delivery system. In the present context the term “drugdelivery system” denotes a pharmaceutical composition (a pharmaceuticalformulation or a dosage form) that upon administration presents theactive substance to the body of a human or an animal. Thus, the term“drug delivery system” embraces plain pharmaceutical compositions suchas, e.g., creams, ointments, liquids, powders, tablets, etc. as well asmore sophisticated formulations such as dressings, devices, templates,smart-gels, grafts etc.

Apart from the active enamel substance, a pharmaceutical composition foruse according to the invention may comprise pharmaceutically orcosmetically acceptable excipients.

A pharmaceutically or cosmetically acceptable excipient is a substancethat is substantially harmless to the individual to which thecomposition is to be administered. Such an excipient normally fulfilsthe requirements given by the national health authorities. Officialpharmacopoeias such as e.g. the British Pharmacopoeia, the United Statesof America Pharmacopoeia and The European Pharmacopoeia set standardsfor pharmaceutically acceptable excipients.

Whether a pharmaceutically acceptable excipient is suitable for use in apharmaceutical composition is generally dependent on which kind ofdosage form is chosen for use for a particular kind of wound. In thefollowing examples of suitable pharmaceutically acceptable excipientsare given for use in different kinds of compositions for use accordingto the invention.

In the following is given a review on relevant pharmaceuticalcompositions for use according to the invention. The review is based onthe particular route of administration. However, it is appreciated thatin those cases where a pharmaceutically acceptable excipient may beemployed in different dosage forms or compositions, the application of aparticular pharmaceutically acceptable excipient is not limited to aparticular dosage form or of a particular function of the excipient.

The choice of pharmaceutically acceptable excipient(s) in a compositionfor use according the invention and the optimum concentration thereofcannot generally be predicted and must be determined on the basis of anexperimental evaluation of the final composition. However, a personskilled in the art of pharmaceutical formulation can find guidance ine.g., “Remington's Pharmaceutical Sciences”, 18th Edition, MackPublishing Company, Easton, 1990.

For application into the tissue defect, the compositions for useaccording to the invention may contain conventionally non-toxicpharmaceutically acceptable carriers and excipients includingmicrospheres and liposomes.

The compositions for use according to the invention include all kinds ofsolid, semi-solid and fluid compositions. Compositions of particularrelevance are e.g. pastes, ointments, hydrophilic ointments, creams,gels, hydrogels, solutions, emulsions, suspensions, powders, films,foams, pads, sponges (e.g. collagen sponges) and transdermal deliverysystems.

The pharmaceutically acceptable excipients may include solvents,buffering agents, preservatives, humectants, chelating agents,antioxidants, stabilizers, emulsifying agents, suspending agents,gel-forming agents, ointment bases, penetration enhancers, perfumes, andskin protective agents.

Examples of solvents are e.g. water, alcohols, vegetable or marine oils(e.g. edible oils like almond oil, castor oil, cacao butter, coconutoil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, peanutoil, poppy seed oil, rape seed oil, sesame oil, soybean oil, sunfloweroil, and tea seed oil), mineral oils, fatty oils, liquid paraffin,polyethylene glycols, propylene glycols, glycerol, liquidpolyalkylsiloxanes, and mixtures thereof.

Examples of buffering agents are e.g. citric acid, acetic acid, tartaricacid, lactic acid, hydrogen phosphoric acid, diethylamine etc.

Suitable examples of preservatives for use in compositions are parabens,such as methyl, ethyl, propyl p-hydroxybenzoate, butylparaben,isobutylparaben, isopropylparaben, potassium sorbate, sorbic acid,benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDMhydantoin, iodopropynyl butylcarbamate, EDTA, benzalconium chloride, andbenzylalcohol, or mixtures of preservatives.

Examples of humectants are glycerin, propylene glycol, sorbitol, lacticacid, urea, and mixtures thereof.

Examples of chelating agents are sodium EDTA and citric acid.

Examples of antioxidants are butylated hydroxy anisole (BHA), ascorbicacid and derivatives thereof, tocopherol and derivatives thereof,cysteine, and mixtures thereof.

Examples of emulsifying agents are naturally occurring gums, e.g. gumacacia or gum tragacanth; naturally occurring phosphatides, e.g. soybeanlecithin; sorbitan monooleate derivatives; wool fats; wool alcohols;sorbitan esters; monoglycerides; fatty alcohols; fatty acid esters (e.g.triglycerides of fatty acids); and mixtures thereof.

Examples of suspending agents are e.g. celluloses and cellulosederivatives such as, e.g., carboxymethyl cellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carraghenan, acacia gum, arabic gum,tragacanth, and mixtures thereof.

Examples of gel bases, viscosity-increasing agents or components whichare able to take up exudate from a wound are: liquid paraffin,polyethylene, fatty oils, colloidal silica or aluminium, zinc soaps,glycerol, propylene glycol, tragacanth, carboxyvinyl polymers,magnesium-aluminium silicates, Carbopol®, hydrophilic polymers such as,e.g. starch or cellulose derivatives such as, e.g.,carboxymethylcellulose, hydroxyethylcellulose and other cellulosederivatives, water-swellable hydrocolloids, carragenans, hyaluronates(e.g. hyaluronate gel optionally containing sodium chloride), andalginates including propylene glycol aginate.

Examples of hydrophobic or water-emulsifying ointment bases areparaffins, vegetable oils, animal fats, synthetic glycerides, waxes,lanolin, and liquid polyalkylsiloxanes.

Examples of hydrophilic ointment bases are solid macrogols (polyethyleneglycols).

Examples of powder components are: alginate, collagen, lactose, powderwhich is able to form a gel when applied to a wound (absorbsliquid/wound exudate). Normally, powders intended for application onlarge open wounds must be sterile and the particles present must bemicronized.

Examples of other excipients are polymers such as carmelose, sodiumcarmelose, hydroxypropylmethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, pectin, xanthan gum, locust bean gum, acaciagum, gelatin, carbomer, emulsifiers like vitamin E, glyceryl stearates,cetanyl glucoside, collagen, carrageenan, hyaluronates and alginates andkitosans.

The compositions mentioned above for topical administration are mostsuitably for application directly into tissue defects or by anyconvenient route of administration.

Compositions which have proved to be of importance in connection withtopical application are those which have tixothropic properties, i.e.the viscosity of the composition is affected e.g. by shaking or stirringso that the viscosity of the composition at the time of administrationcan be reduced and when the composition has been applied, the viscosityincreases so that the composition remains at the application site.

Suitable compositions for use according to the invention may also bepresented in the form of suspensions, emulsions or dispersions. Suchcompositions contain the active enamel substance in admixture with adispersing or wetting agent, suspending agent, and/or one or morepreservatives and other pharmaceutically acceptable excipients. Suchcompositions may also be suitable for use in the delivery of the activeenamel substance to e.g. an intact or damaged tissue with a cavity likedefect.

Suitable dispersing or wetting agents are, e.g., naturally occurringphosphatides, e.g., lecithin, or soybean lecithin; condensation productsof ethylene oxide with e.g. a fatty acid, a long chain aliphaticalcohol, or a partial ester derived from fatty acids and a hexitol or ahexitol anhydride, e.g. polyoxyethylene stearate, polyoxyethylenesorbitol monooleate, polyoxyethylene sorbitan monooleate, etc.

Suitable suspending agents are, e.g., naturally occurring gums such as,e.g., gum acacia, xanthan gum, or gum tragacanth; celluloses such as,e.g., sodium carboxymethylcellulose, microcrystalline cellulose (e.g.Avicel® RC 591, methylcellulose); alginates and kitosans such as, e.g.,sodium alginate, etc.

Suitable examples of preservatives for use in compositions according tothe invention are the same as those mentioned above.

In a pharmaceutical composition for use according to the invention onskin or mucosa, an active enamel substance is generally present in aconcentration ranging from about 0.01% to about 99.9% w/w. The amount ofcomposition applied will normally result in an amount of total proteinper cm² wound/tissue-defect area, corresponding to from about 0.01mg/cm² to about 20 mg/cm² such as from about 0.1 mg/cm² to about 15mg/cm².

The amount applied of the composition depends on the concentration ofthe active enamel substance in the composition and of the release rateof the active enamel substance from the composition, but is generally ina range corresponding to at the most about 15–20 mg/cm².

In those cases where the active enamel substance is administered in theform of a fluid composition, the concentration of the active enamelsubstance in the composition is in a range corresponding to from about0.1 to about 50 mg/ml. Higher formulation/dosages are in some casesdesirable and can also be obtained such as of at least about 50 mg/ml.

The concentration of the active enamel substance in a pharmaceuticalcomposition depends on the specific enamel substance, its potency, theseverity of the tissue loss or defect to be prevented or treated, andthe age and condition of the patient. Methods applicable to selectingrelevant concentrations of the active enamel substance in thepharmaceutical composition are well known to a person skilled in the artand may be performed according to established guidelines for goodclinical practice (GCP) or Investigational New Drug Exemption (“IND”)regulations as described in e.g. International Standard ISO/DIS 14155Clinical investigation of medical devices, 1994 and ICH (InternationalCommittee for Harmonisation): Harmonised tripartite guideline for goodclinical practice, Brookwood Medical Publications, Ltd, Surrey, UK,1996. A person skilled in the art would, by use of the methods describedin standard textbooks, guidelines and regulations as described above aswell as common general knowledge within the field, be able to select theexact dosage regimen to be implemented for any active enamel substanceand/or selected other active substances and dosage form using merelyroutine experimentation procedures.

As will be understood, details and particulars concerning the use of anactive enamel substance for the induction and stimulation of connectivetissue cell invasion, proliferation and growth will be the same as oranalogous to the details and particulars concerning the other useaspects (anti scarring and contraction resistance) and the methodaspects discussed above, and this means that wherever appropriate, thestatements above concerning an active enamel substance, a preparationcontaining an active enamel substance, a pharmaceutical compositioncontaining an active enamel substance, preparation of i) an activeenamel substance, ii) a preparation containing an active enamelsubstance, iii) a pharmaceutical composition containing an active enamelsubstance, as well as improved properties and uses apply mutatismutandis to all aspects of the invention.

The observation, that enamel matrix is formed and temporarily presentduring root and root cementum formation can explain how application ofenamel matrix, enamel matrix derivatives and/or enamel matrix proteinspromotes the regeneration of periodontal tissue.

However, the observation underlying the present invention that enamelmatrix, enamel matrix derivatives and/or enamel matrix proteins alsohave a positive effect on invasion, proliferation and growth ofconnective tissue cells is very surprising. The same applies to theobservations with respect to the reduced scarring and contractionobserved in treated defects. As demonstrated in the experimental sectionherein, the active enamel substance aggregates and revitalises radiatedfibroblasts cells and initiates fibroblast cell migration, replicationand growth.

Experiments

The therapeutic and/or prophylactic activity of active enamel substancesmay of course be evidenced by in vivo tests, using experimental animalsor humans. However, an indication of the efficacy and/or activity ofenamel matrix, enamel matrix derivatives and/or enamel matrix proteinscan be obtained by performing relatively simple in vitro tests such as,e.g., tests involving cell cultures.

Furthermore, there are several parameters that may be employed in orderto evaluate a wound healing effect. These include:

-   -   Ultra-sound analysis of treated tissues    -   Magnet resonance imaging (MR)    -   Histopathology/cytology (microscopic evaluation of wound tissues        and fluids)    -   Scintigraphy (radionuclide imaging of wound tissue)

EXAMPLE 1

Investigation of growth behaviour of dermal fibroblasts cultured in thepresence of enamel matrix derivative.

The purpose of this example is to show the capacity of enamel matrixderivative to induce and stimulate dermal fibroblast cell attachment,replication and growth.

Cell Isolation and Culture Conditions

Human dermal fibroblasts, obtained from ATTC, were cultured from healthydermal tissues from young healthy volunteering individuals. Cultureswere maintained in DMEM with 10% foetal bovine serum. EMD,EMDOGAIN®(BIORA AB, Sweden), was added prior to commencement of cellcultures, by coating charged plastic culture dishes with a 0.5 mg/ml EMDsolution in 0.1% HAc in PBS overnight. In addition, the medium wassupplemented with 100 μg EMD per milliliter. The EMD concentrations werechosen on the basis of pilot experiments with cultured cells indicatingthat optimal growth occurred at these values, and that cell growth didnot benefit from further addition of EMD. There were no changes of mediaduring the five to seven day observation period of this study. Allexperiments commenced with 50,000 cells per milliliter of culturemedium.

Cell Attachment

To assess the cell attachment rate during the first four hours afterseeding, 100,000 cells were cultured on EMD coated surfaces for 30, 60,120 or 240 minutes before the cultures were vigorously washed with PBSto remove all unattached cells. The washing solution was centrifuged andthe numbers of unattached cells were analysed using a Bürker chamber.The attached cells were then removed from the surface by trypsinisationand counted in the same way for control. Uncoated dishes were used asnegative control.

Cell Culture Densities

Cells were seeded and maintained in cultures with or without EMD for 24,48, 72, 96 or 120 hours. Cultures were then carefully washed with PBSand the number of attached cells per square millimeter was calculated inthe microscope using a fixed grid.

Cell Metabolism

Cells were cultured for 24, 48, 72, 96, 120 or 144 hours and then givena 4-hour pulse of 50 μCi [³⁵S]methionine (Cell culture grade, AmershamPharmacia Biotech). The cultures were then washed with PBS and the cellswere removed by trypsinisation. The cells were then washed again,centrifuged and 200 μl of each cell pellet were dissolved in UniverSol™liquid scintillation cocktail (ICN Biomedicals Inc.) and counted twotimes 300 seconds in a Packard Tricarb scintillation counter.

Nucleic Acid Synthesis

Nucleic acid synthesis in cells cultures 24, 48, 72 or 96 hours wasassessed by colorimetric analysis at 370 nm following a 4-hour pulsewith BrdU using the Boehringer Mannheim Cell PROLIFERATION ELISA, BRDUKIT® (Cat. No. 164229). During the pulse, the pyrimidine analog BrdU wasincorporated in place of thymidine into the newly synthesized DNA ofproliferating cells. At the end of the pulse the cells were washed,fixed and denatured and the amount of incorporated BrdU was measured byELISA utilizing an anti-BrdU peroxidase conjugated antibody.

Results

The experiments showed that fibroblast cell attachment rate during thefirst hours after seeding is nearly five times more efficient when thesurface of the culture dish is coated with EMD (FIG. 1). Cell density inthe cultures increased faster when EMD was present (FIG. 2). The generaltrend was that cultures growing in the presence of EMD got a one-daylead, reaching confluence after four days in culture, one day ahead ofthe control cells. The metabolic rate of fibroblast cells also increasedon a per cell basis in cultures seeded on EMD as compared to controlcultures (FIG. 3). This increase in [³⁵S]methionine utilisation washigher than the observed change in growth rate alone.

In pulse-chase experiments with the thymidine analogue bromodeoxyuridine(BrdU) fibroblast cell cultures showed an increased DNA synthesis in thepresence of EMD (FIG. 4). The results prove that EMD stimulatesfibroblast proliferation and differentiation. The fibroblasts in theexperiment attached to the dishes and grew quicker than those in theuncoated dishes, their metabolism was enhanced and they most probablyexpressed cell-specific proteins.

EXAMPLE 2

Investigation of recovery of fibroblast cells exposed to ionisingradiation by application of enamel matrix derivative.

The purpose of this example is to show the ability of enamel matrixderivative to revitalise fibroblast cells that have been exposed toionising radiation.

Cell Isolation and Culture Conditions

Human dermal fibroblasts, obtained from ATTC, were cultured from healthydermal tissues from young healthy volunteering individuals. Cultureswere maintained in DMEM with 10% foetal bovine serum. EMD was addedprior to commencement of cell cultures, by coating charged plasticculture dishes with a 0.5 mg/ml EMD solution in 0.10% HAc in PBSovernight. In addition, the medium was supplemented with 100 μg of EMDper milliliter. The EMD concentrations were chosen on the basis of pilotexperiments with cultured cells indicating that optimal growth occurredat these values, and that cell growth did not benefit from furtheraddition of EMD. There were no changes of media during the five to sevenday observation period of this study. All experiments commenced with50,000 cells per milliliter of culture medium.

Radiation Therapy

Confluent cultures of dermal fibroblast cells, cultured without EMD,received a gamma radiation dosage of 0, 1, 2, 5 10, 15 and 20 Grey.Immediately after radiation therapy, the cultures were split in two bytrypsination and seeding according to standard operating procedures. Oneof the parallels was then cultured with 100 μg/ml EMD present in themedium, whereas the other was cultured without EMD as unstimulatedcontrol.

Cell Culture Densities

Cells were seeded and maintained in cultures with or without EMD for 24,48, 72, 96 or 120 hours. Cultures were then carefully washed with PBSand the number of attached cells per square millimeter was calculated inthe microscope using a fixed grid.

Results

Radiated cells growing in the presence of EMD increased their numbertwice as fast as unstimulated cultures (FIGS. 5A and 5B). This trend wastrue for all cultures, even though in general the growth potential ofthe cultured dermal fibroblasts decreased with increasing radiationdosages. The results demonstrate that EMD can revitalise radiatedcultured dermal fibroblast cells, and that the presence of EMD proteinsstimulates proliferation and growth of these cells also when basiccellular functions are impaired by radiation damage.

EXAMPLE 3

Investigation of soft tissue defect fill and prevention of scarring andtissue contraction after surgical removal of a breast tumour followed byradiation therapy.

The purpose of this example is to show the influence of active enamelsubstances on improvement of soft tissue defect fill and reduced tissuecontraction after cytoreductive surgery and radiation therapy.

An active enamel substance may be applied either directly into a softtissue defect prior to suturing or it may be injected into the woundcavity after suturing. The volume/amount of active enamel substancesapplied will differ from case to case and tissue to tissue, but mostlythe therapy will aim at replacing the volume of the lost tissue.However, in cases where an increase or decrease of the volume of atissue or body part, e.g. a breast, is desirable, the enamel matrix,enamel matrix derivatives and/or enamel matrix proteins may be appliedin surplus or deficit to acquire the desired outcome. The active enamelsubstance may be used as such or may be used in a suitable preparationor pharmaceutical composition.

A patient with a diagnosed adenocarcinoma in the breast is submitted forsurgical removal of the tumor according to standard aesthetic andsurgical procedures. After resection of the tumor tissue, the mass ofthe removed tissue is estimated to about 5 ml. The wound cavity in thebreast is then half closed by suturing at the incision, carefullyavoiding pull, tension or contraction in the walls of the wound cavity.The wound cavity is then thoroughly rinsed with sterile saline to removeblood clots, cell debris and damaged tissue. After thoroughly drainingthe saline from the wound cavity, EMD in PGA in the form of EMDOGAIN®Gel (BIORA AB, Sweden) is injected into the wound cavity so that thewhole of the cavity is filled (volume>5 ml). Finally, the wound cavityis closed by sutures at the incision site, taking care that theEMDOGAIN® Gel (BIORA AB, Sweden) remains in situ. No draining device isapplied, and standard postoperative procedures and wound care areapplied.

The patient is allowed to recover from surgery for two weeks beforehe/she is submitted to adjuvant radiation therapy of the treated breast.Prior to the radiation therapy, the wound healing is assessed byclinical examination, ultrasound and imaging in order to monitorconnective tissue growth in the tissue defect. Clinical pictures andmeasurements of the size and location of the healing defect arerecorded.

To ensure total eradication of malignant cells from the breast, thepatient is submitted to local radiation therapy. The radiation dosagedepends on the phenotype of the malignant cells (assessed by histologyon tissue removed during surgery), the size of the breast and thecondition of the patient (age, weight, systemic diseases etc.). Thetypical dosage ranges between 0.5 and 20 Gray. If considered necessary,radiation therapy is repeated until the patient is declared free fromprimary tumour cells.

Following the first radiation therapy, the patient is monitored everyweek the first four weeks and then every month the next six months, forconnective tissue wound fill, tissue contractions and appearance. Thehealing process and cell invasion into the tissue defect is monitored byclinical measurements, palpation, photography, and/or ultra soundimaging. The patient is asked to fill in a questionnaire regardingpostoperative discomfort and the progression of the healing. Ifpossible, aspiration biopsies are obtained from the healing breast toassess the type and quantity of cellular ingrowth in the defect.

In the frequent normal cases without the use of EMD, the wound cavitystarts to contract after the first radiation therapy. This is due to theremoval of proliferating fibroblasts from the wound by radiation,leaving a cell poor dense connective tissue that shrinks as the woundorganises. At six months, the wound contraction often is so severe thatthe patients prefer to have most of the breast removed and replaced bysilicon inlays. Thus, conservative surgery in breasts scheduled forradiation therapy often fails. However, by application of EMD into thewound cavity prior to radiation therapy, a wound fill comprising cellrich, loose connective tissue that is less sensitive to radiationinduced contraction is achieved. By reducing contraction in thesewounds, the post radiation wound healing is improved and the need foradditional corrective surgery and/or prosthesis treatment issignificantly reduced.

LIST OF REFERENCES

-   1. Hammarström et al., 1997, Journal of Clinical Periodontology 24,    658–668-   2. Lyngstadaas et al., 2000, Journal of Clinical Periodontology 27,    1–8-   3. Ten Cate: Oral Histology, 1994;-   4. Robinson: Eur. J. Oral Science, January 1998, 106 Suppl. 1:282–91-   5. Janson, J-C & Rydén, L. (Eds.), Protein purification, VCH    Publishers 1989-   6. Fincham et al. in J. Struct. Biol. 1994 March–April; 112(2):    103–9 and in 3. Struct. Biol. 1995 July–August; 115(1): 50–9)-   7. “Remington's Pharmaceutical Sciences” and “Encyclopedia of    Pharmaceutical Technology”, edited by Swarbrick, J. & J. C. Boylan,    Marcel Dekker, Inc., New York, 1988-   8. “Remington's Pharmaceutical Sciences”, 18th Edition, Mack    Publishing Company, Easton, 1990-   9. International Standard ISO/DIS 14155 Clinical investigation of    medical devices, 1994-   10. Harmonised tripartite guideline for good clinical practice,    Brookwood Medical Publications, Ltd, Surrey, UK, 1996-   11. Harris, ELV & Angal, S., Protein purification methods—A    practical approach, IRL Press, Oxford 1990-   12. Sambrook, J. et al.: Molecular Cloning, Cold Spring Harbor    Laboratory Press, 1989-   13. Gestrelius S, Lyngstadaas SP, Hammarstrøm L.    Emdogain—periodontal regeneration based on biomimicry. Clin Oral    Invest 4:120–125, 2000

1. A pharmaceutical or cosmetic composition for filling a wound cavityand/or tissue defect resulting from a procedure and/or trauma followingcytoreductive surgery, said composition comprising an active enamelsubstance, wherein the active enamel substance is enamel matrix, enamelmatrix derivatives, or enamel matrix proteins.
 2. The compositionaccording to claim 1, wherein said composition stimulates cellularneogenesis in said wound cavity and/or tissue defect.
 3. The compositionaccording to claim 1, wherein said composition stimulates cellularproliferation, differentiation and/or maturation in said wound cavityand/or tissue defect.
 4. The composition according to claim 3, whereinthe stimulation is cell-type specific.
 5. The composition according toclaim 4, wherein the stimulation is cell-type specific for a cell frommesodermal and/or endodermal origin.
 6. The composition according toclaim 1, wherein the cavity or defect is caused by cytoreductivesurgery.
 7. The composition according to claim 1, wherein the cavity ordefect is at least partly caused by radiation therapy.
 8. Thecomposition according to claim 1, wherein the cavity or defect is causedby a bodily injury, infection or trauma.
 9. The composition according toclaim 1, wherein the cavity or defect is caused by the surgical removalof a tumor selected from the group consisting of mammalian neoplasm,neck and head cancer, abdominal cancer, ovarian cancer, breast cancerand skin cancer.
 10. The composition according to claim 1, wherein thecavity or defect is caused by the surgical removal of a breast tumor.11. The composition according to claim 1, wherein the active enamelsubstance is selected from the group consisting of enamelins,amelogenins, non-amelogenins, proline-rich non-amelogenins, tuftelins,and mixtures thereof.
 12. The composition according to claim 1, whereinthe active enamel substance has a molecular weight that does not exceedabout 120 kDa as determined by SDS PAGE electrophoresis.
 13. Thecomposition according to claim 1, wherein the active enamel substancehas a molecular weight between about 60 kDa and 100 kDa as determined bySDS PAGE electrophoresis.
 14. The composition according to claim 1,wherein the composition comprises a mixture of active enamel substanceswith different molecular weights.
 15. The composition according to claim1, wherein the active enamel substance comprises at least two substancesselected from the group consisting of amelogenins, proline-richnon-amelogenins, tuftelins, tuft proteins, serum proteins, salivaryproteins, ameloblastin, and sheathlin.
 16. The composition according toclaim 1, wherein the active enamel substance has a molecular weight ofup to about 40,000.
 17. The composition according to claim 1, whereinthe active enamel substance has a molecular weight of between about5,000 and about 25,000.
 18. The composition according to claim 1,wherein the major part of the active enamel substance has a molecularweight of about 20 kDa.
 19. The composition according to claim 1,wherein at least a part of the active enamel substance is in the form ofaggregates or after application in vivo is capable of formingaggregates.
 20. The composition according to claim 19, wherein theaggregates have a particle size of from about 20 nm to about 1 μm. 21.The composition according to claim 1, wherein the active enamelsubstance has a protein content in the composition in a range of fromabout 0.05% w/w to 100% w/w.
 22. The composition according to claim 1,wherein the active enamel substance has a protein content in thecomposition in a range of from about 5% w/w to 99% w/w.
 23. Thecomposition according to claim 1, wherein the active enamel substancehas a protein content in the composition in a range of from about 10–95%w/w.
 24. The composition according to claim 1, wherein the active enamelsubstance has a protein content in the composition in a range of fromabout 15–90% w/w.
 25. The composition according to claim 1, wherein theactive enamel substance has a protein content in the composition in arange of from about 20–90% w/w.
 26. The composition according to claim1, wherein the active enamel substance has a protein content in thecomposition in a range of from about 30–90% w/w.
 27. The compositionaccording to claim 1, wherein the active enamel substance has a proteincontent in the composition in a range of from about 40–85% w/w.
 28. Thecomposition according to claim 1, wherein the active enamel substancehas a protein content in the composition in a range of from about 50–80%w/w.
 29. The composition according to claim 1, wherein the active enamelsubstance has a protein content in the composition in a range of fromabout 60–70% w/w.
 30. The composition according to claim 1, wherein theactive enamel substance has a protein content in the composition in arange of from about 70–90% w/w.
 31. The composition according to claim1, wherein the pharmaceutical composition further comprises apharmaceutically acceptable excipient.
 32. The composition according toclaim 31, wherein the pharmaceutically acceptable excipient is propyleneglycol alginate.
 33. The composition according to claim 1, wherein thepharmaceutical composition comprises 68 mg of enamel matrix protein and1 ml of vehicle solution.
 34. A method for the treatment and/orreduction of scarring and/or wound contraction comprising administeringan effective amount of a pharmaceutical or cosmetic composition forfilling a wound cavity and/or tissue defect resulting from a procedureand/or trauma following cytoreductive surgery such that the scarringand/or wound contraction is treated and/or reduced, said compositioncomprising an active enamel substance, wherein the active enamelsubstance is enamel matrix, enamel matrix derivatives, or enamel matrixproteins.
 35. A method according to claim 34, wherein the tissue isselected from the group consisting of breast tissue, lip tissue,abdominal wall tissue, facial tissue, neck tissue, tissue of theextremities, soft tissue or muscle tissue.
 36. The method according toclaim 34, wherein scarring and contraction that is reduced or treatedaffects a normal function of a breast, lip, abdominal wall, face, neck,and/or extremities.
 37. The method according to claim 34, whereinscarring and contraction is reduced or treated so as to affect theappearance of the patient.
 38. The method according to claim 34, whereinthe scarring and contraction is at least partly caused by infection,malnutrition, inflammation, systemic diseases, and/or pathologicalconditions.
 39. A method for promoting the filling of a wound cavityand/or tissue defect in a patient, said cavity and/or defect resultingfrom a procedure and/or trauma following cytoreductive surgery, whereinan effective amount of a pharmaceutical composition comprising an activeenamel substance is administered to said wound cavity and/or tissuedefect thereby filling the wound cavity and/or tissue defect, andwherein the active enamel substance is enamel matrix, enamel matrixderivatives, or enamel matrix proteins.
 40. A method for promoting there-filling of a wound cavity and/or tissue defect in a patient resultingfrom a procedure and/or trauma following cytoreductive surgery, whereinan effective amount of a pharmaceutical composition comprising an activeenamel substance is administered to said wound cavity and/or tissuedefect thereby re-filling the wound cavity and/or tissue defect and,wherein the active enamel substance is enamel matrix, enamel matrixderivatives, or enamel matrix proteins.
 41. The method according to anyof claim 39 or 40, wherein said administered composition stimulatescellular neogenesis of cells with mesodermal and/or endodermal origin insaid wound cavity and/or tissue defect.
 42. The method according to anyof claim 39 or 40, wherein the cavity or defect is caused bycytoreductive surgery and/or wherein the cavity or defect is at leastpartly caused by radiation therapy.
 43. The method according to claim42, wherein said cavity or defect is caused by the surgical removal of atumor selected from the group consisting of mammalian neoplasm, neck andhead cancer, abdominal cancer, ovarian cancer, breast cancer and skincancer.
 44. The composition of claim 33, wherein the vehicle ispropylene glycol alginate.